Time- and sex-dependent delayed effects of acute radiation exposure manifest via miRNA dysregulation.

The detrimental effects of high-dose ionizing radiation on human health are well-known, but the influence of sex differences on the delayed effects of acute radiation exposure (DEARE) remains unclear. Here, we conducted six-month animal experiments using escalating radiation doses (7-9 Gy) on male and female C57BL/6 mice. The results show that female mice exhibited greater resistance to radiation, showing increased survival at six months post-total body irradiation. LD50/30 (lethal dose expected to cause 50% lethality in 30 days) for female mice is 8.08 Gy, while for male mice it is 7.76 Gy. DEARE causes time- and sex-dependent dysregulation of microRNA expression, processing enzymes, and the HOTAIR regulatory pathway. Differential regulation of molecular patterns associated with growth, development, apoptosis, and cancer is also observed in male and female mice. These findings shed light on the molecular basis of age and sex differences in DEARE response and emphasize the importance of personalized medicine for mitigating radiation-induced injuries and diseases.

PEGylated thrombopoietin mimetic, JNJ­26366821 a novel prophylactic radiation countermeasure for acute radiation injury.

Thrombopoietin (TPO) is the primary regulator of platelet generation and a stimulator of multilineage hematopoietic recovery following exposure to total body irradiation (TBI). JNJ­26366821, a novel PEGylated TPO mimetic peptide, stimulates platelet production without developing neutralizing antibodies or causing any adverse effects. Administration of a single dose of JNJ­26366821 demonstrated its efficacy as a prophylactic countermeasure in various mouse strains (males CD2F1, C3H/HeN, and male and female C57BL/6J) exposed to Co-60 gamma TBI. A dose dependent survival efficacy of JNJ­26366821 (- 24 h) was identified in male CD2F1 mice exposed to a supralethal dose of radiation. A single dose of JNJ­26366821 administered 24, 12, or 2 h pre-radiation resulted in 100% survival from a lethal dose of TBI with a dose reduction factor of 1.36. There was significantly accelerated recovery from radiation-induced peripheral blood neutropenia and thrombocytopenia in animals pre-treated with JNJ­26366821. The drug also increased bone marrow cellularity and megakaryocytes, accelerated multi-lineage hematopoietic recovery, and alleviated radiation-induced soluble markers of bone marrow aplasia and endothelial damage. These results indicate that JNJ­26366821 is a promising prophylactic radiation countermeasure for hematopoietic acute radiation syndrome with a broad window for medical management in a radiological or nuclear event.

PrC-210 Protects against Radiation-Induced Hematopoietic and Intestinal Injury in Mice and Reduces Oxidative Stress.

The development of safe, orally available, and effective prophylactic countermeasures to protect our warfighters is an unmet need because there is no such FDA-approved countermeasure available for use. Th 1-Propanethiol, 3-(methylamino)-2-((methylamino)methyl) (PrC-210), a synthetic small molecule, is a member of a new family of aminothiols designed to reduce toxicity while scavenging reactive oxygen species (ROS). Our study investigated the protective role of a single oral administration of PrC-210 against radiation-induced hematopoietic and intestinal injury in mice. Pre-treatment with PrC-210 significantly improved the survival of mice exposed to a lethal dose of radiation. Our findings indicated that the radioprotective properties of PrC-210 are achieved by accelerating the recovery of the hematopoietic system, stimulating bone marrow progenitor cells, and ameliorating additional biomarkers of hematopoietic injury. PrC-210 pre-treatment reduced intestinal injury in mice exposed to a lethal dose of radiation by restoring jejunal crypts and villi, reducing translocation of bacteria to the spleen, maintaining citrulline levels, and reducing the sepsis marker serum amyloid A (SAA) in serum. Finally, PrC-210 pre-treatment led to a significant reduction (~10 fold) of Nos2 expression (inducible nitric oxide) in the spleen and decreased oxidative stress by enhancing the antioxidant defense system. These data support the further development of PrC-210 to receive approval from the FDA to protect warfighters and first responders from exposure to the harmful effects of ionizing radiation.

CDX-301 prevents radiation-induced dysregulation of miRNA expression and biogenesis.

Risks of radiation exposure necessitate the development of radioprophylactic drugs. We have reported the efficacy of CDX-301, a recombinantly developed human protein form of Fms-related tyrosine kinase 3 ligand (Flt3L), as a radioprophylactic and radiomitigatory agent. Here, we performed global microRNA profiling to further understand the mechanism of action of CDX-301. We find that CDX-301 administration 24 h prior to total body irradiation prevents radiation-induced dysregulation of microRNA biogenesis and expression in murine serum and spleen samples in a time- and tissue-dependent manner. Further analysis shows that activation of the HOTAIR regulatory pathway has a prominent function in radiation-induced injury responses, which is inhibited by pre-treatment with CDX-301. Moreover, CDX-301 attenuates radiation-induced dysregulation of several cellular functions such as inflammatory and immune responses. In corroboration, we also find that pre-treatment with CDX-301 restores the expression of bone marrow aplasia markers and inflammatory cytokines and growth factors, as well as the expression of genes associated with MAP kinase and TGF-ß pathways that are altered by radiation. Our findings provide new insights into CDX-301-mediated molecular and cellular mechanisms and point to a possible novel radioprotective drug for the prevention of irradiation-induced injury and hematopoietic acute radiation syndrome.

Pre-Administration of PLX-R18 Cells Protects Mice from Radiation-Induced Hematopoietic Failure and Lethality.

Acute Radiation Syndrome (ARS) is a syndrome involving damage to multiple organs caused by exposure to a high dose of ionizing radiation over a short period of time; even low doses of radiation damage the radiosensitive hematopoietic system and causes H-ARS. PLacenta eXpanded (PLX)-R18 is a 3D-expanded placenta-derived stromal cell product designated for the treatment of hematological disorders. These cells have been shown in vitro to secrete hematopoietic proteins, to stimulate colony formation, and to induce bone marrow migration. Previous studies in mice showed that PLX-R18 cells responded to radiation-induced hematopoietic failure by transiently secreting hematopoiesis related proteins to enhance reconstitution of the hematopoietic system. We assessed the potential effect of prophylactic PLX-R18 treatment on H-ARS. PLX-R18 cells were administered intramuscularly to C57BL/6 mice, −1 and 3 days after (LD70/30) total body irradiation. PLX R18 treatment significantly increased survival after irradiation (p < 0.0005). In addition, peripheral blood and bone marrow (BM) cellularity were monitored at several time points up to 30 days. PLX-R18 treatment significantly increased the number of colony-forming hematopoietic progenitors in the femoral BM and significantly raised peripheral blood cellularity. PLX-R18 administration attenuated biomarkers of bone marrow aplasia (EPO, FLT3L), sepsis (SAA), and systemic inflammation (sP-selectin and E-selectin) and attenuated radiation-induced inflammatory cytokines/chemokines and growth factors, including G-CSF, MIP-1a, MIP-1b, IL-2, IL-6 and MCP-1, In addition, PLX-R18 also ameliorated radiation-induced upregulation of pAKT. Taken together, prophylactic PLX-R18 administration may serve as a protection measure, mitigating bone marrow failure symptoms and systemic inflammation in the H-ARS model.

Mitigation of total body irradiation-induced mortality and hematopoietic injury of mice by a thrombopoietin mimetic (JNJ-26366821).

The threat of a nuclear attack has increased in recent years highlighting the benefit of developing additional therapies for the treatment of victims suffering from Acute Radiation Syndrome (ARS). In this work, we evaluated the impact of a PEGylated thrombopoietin mimetic peptide, JNJ-26366821, on the mortality and hematopoietic effects associated with ARS in mice exposed to lethal doses of total body irradiation (TBI). JNJ-26366821 was efficacious as a mitigator of mortality and thrombocytopenia associated with ARS in both CD2F1 and C57BL/6 mice exposed to TBI from a cobalt-60 gamma-ray source. Single administration of doses ranging from 0.3 to 1 mg/kg, given 4, 8, 12 or 24 h post-TBI (LD70 dose) increased survival by 30-90% as compared to saline control treatment. At the conclusion of the 30-day study, significant increases in bone marrow colony forming units and megakaryocytes were observed in animals administered JNJ-26366821 compared to those administered saline. In addition, enhanced recovery of FLT3-L levels was observed in JNJ-26366821-treated animals. Probit analysis of survival in the JNJ-26366821- and saline-treated cohorts revealed a dose reduction factor of 1.113 and significant increases in survival for up to 6 months following irradiation. These results support the potential use of JNJ-26366821 as a medical countermeasure for treatment of acute TBI exposure in case of a radiological/nuclear event when administered from 4 to 24 h post-TBI.

Corrigendum: Gamma Tocotrienol Protects Mice From Targeted Thoracic Radiation Injury.

[This corrects the article DOI: 10.3389/fphar.2020.587970.].

Gamma Tocotrienol Protects Mice From Targeted Thoracic Radiation Injury.

Radiation injury will result in multiorgan dysfuntion leading to multiorgan failure. In addition to many factors such as radiation dose, dose rate, the severity of the injury will also depend on organ systems which are exposed. Here, we report the protective property of gamma tocotrienol (GT3) in total as well as partial body irradiation (PBI) model in C3H/HeN male mice. We have carried out PBI by targeting thoracic region (lung-PBI) using Small Animal Radiation Research Platform, an X-ray irradiator with capabilities of an image guided irradiation with a variable collimator with minimized exposure to non-targeted tissues and organs. Precise and accurate irradiation of lungs was carried out at either 14 or 16 Gy at an approximate dose rate of 2.6 Gy/min. Though a low throughput model, it is amenable to change the field size on the spot. No damage to other non-targeted organs was observed in histopathological evaluation. There was no significant change in peripheral blood counts of irradiated mice in comparison to naïve mice. Femoral bone marrow cells had no damage in irradiated mice. As expected, damage to the targeted tissue was observed in the histopathological evaluation and non-targeted tissue was found normal. Regeneration and increase of cellularity and megakaryocytes on GT3 treatment was compared to significant loss of cellularity in saline group. Peak alveolitis was observed on day 14 post-PBI and protection from alveolitis by GT3 was noted. In irradiated lung tissue, thirty proteins were found to be differentially expressed but modulated by GT3 to reverse the effects of irradiation. We propose that possible mode of action of GT3 could be Angiopoietin 2-Tie2 pathway leading to AKT/ERK pathways resulting in disruption in cell survival/angiogenesis.

Delayed effects of acute whole body lethal radiation exposure in mice pre-treated with BBT-059.

The threat of nuclear exposure is heightened and it is imperative to identify potential countermeasures for acute radiation syndrome. Currently no countermeasures have been approved for prophylactic administration. Effective countermeasures should function to increase survival in the short term as well as to increase the overall prognosis of an exposed individual long term. Here we describe the use of a promising radiation countermeasure, BBT-059, and the results of a long term mouse study (up to 12 months) in the male CD2F1 strain using 60Co gamma irradiation (~0.6 Gy/min, 7.5-12.5 Gy). We report the dose reduction factor of 1.28 for BBT-059 (0.3 mg/kg) compared to control administered 24 h prior to irradiation. In the long term study animals showed accelerated recovery in peripheral blood cell counts, bone marrow colony forming units, sternal cellularity and megakaryocyte numbers in drug treated mice compared to formulation buffer. In addition, increased senescence was observed in the kidneys of animals administered control or drug and exposed to the highest doses of radiation. Decreased levels of E-cadherin, LaminB1 and increased levels of Cyc-D and p21 in spleen lysates were observed in animals administered control. Taken together the results indicate a high level of protection following BBT-059 administration in mice exposed to lethal and supralethal doses of total body gamma-radiation.

microRNA and Metabolite Signatures Linked to Early Consequences of Lethal Radiation.

Lethal total body irradiation (TBI) triggers multifactorial health issues in a potentially short time frame. Hence, early signatures of TBI would be of great clinical value. Our study aimed to interrogate microRNA (miRNA) and metabolites, two biomolecules available in blood serum, in order to comprehend the immediate impacts of TBI. Mice were exposed to a lethal dose (9.75 Gy) of Cobalt-60 gamma radiation and euthanized at four time points, namely, days 1, 3, 7 and 9 post-TBI. Serum miRNA libraries were sequenced using the Illumina small RNA sequencing protocol, and metabolites were screened using a mass spectrometer. The degree of early impacts of irradiation was underscored by the large number of miRNAs and metabolites that became significantly expressed during the Early phase (day 0 and 1 post-TBI). Radiation-induced inflammatory markers for bone marrow aplasia and pro-sepsis markers showed early elevation with longitudinal increment. Functional analysis integrating miRNA-protein-metabolites revealed inflammation as the overarching host response to lethal TBI. Early activation of the network linked to the synthesis of reactive oxygen species was associated with the escalated regulation of the fatty acid metabolism network. In conclusion, we assembled a list of time-informed critical markers and mechanisms of significant translational potential in the context of a radiation exposure event.

Mitochondrial Degeneration and Autophagy Associated With Delayed Effects of Radiation in the Mouse Brain.

Mitochondria are linked with various radiation responses, including mitophagy, genomic instability, apoptosis, and the bystander effect. Mitochondria play an important role in preserving cellular homeostasis during stress responses, and dysfunction in mitochondrial contributes to aging, carcinogenesis and neurologic diseases. In this study, we have investigated the mitochondrial degeneration and autophagy in the hippocampal region of brains from mice administered with BBT-059, a long-acting interleukin-11 analog, or its formulation buffer 24 h prior to irradiation at different radiation doses collected at 6 and 12 months post-irradiation. The results demonstrated a higher number of degenerating mitochondria in 12 Gy BBT-059 treated mice after 6 months and 11.5 Gy BBT-059 treated mice after 12 months as compared to the age-matched naïve (non-irradiated control animals). Apg5l, Lc3b and Sqstm1 markers were used to analyze the autophagy in the brain, however only the Sqstm1 marker exhibited significantly reduced expression after 12 months in 11.5 Gy BBT-059 treated mice as compared to naïve. Immunohistochemistry (IHC) results of Bcl2 also demonstrated a decrease in expression after 12 months in 11.5 Gy BBT-059 treated mice as compared to other groups. In conclusion, our results demonstrated that higher doses of ionizing radiation (IR) can cause persistent upregulation of mitochondrial degeneration. Reduced levels of Sqstm1 and Bcl2 can lead to intensive autophagy which can lead to degradation of cellular structure.

Proteomic Changes in Mouse Spleen after Radiation-Induced Injury and its Modulation by Gamma-Tocotrienol.

Gamma-tocotrienol (GT3), a naturally occurring vitamin E isomer, a promising radioprotector, has been shown to protect mice against radiation-induced hematopoietic and gastrointestinal injuries. We analyzed changes in protein expression profiles of spleen tissue after GT3 treatment in mice exposed to gamma radiation to gain insights into the molecular mechanism of radioprotective efficacy. Male CD2F1 mice, 12-to-14 weeks old, were treated with either vehicle or GT3 at 24 h prior to 7 Gy total-body irradiation. Nonirradiated vehicle, nonirradiated GT3 and age-matched naïve animals were used as controls. Blood and tissues were harvested on days 0, 1, 2, 4, 7, 10 and 14 postirradiation. High-resolution mass-spectrometry-based radioproteomics was used to identify differentially expressed proteins in spleen tissue with or without drug treatment. Subsequent bioinformatic analyses helped delineate molecular markers of biological pathways and networks regulating the cellular radiation responses in spleen. Our results show a robust alteration in spleen proteomic profiles including upregulation of the Wnt signaling pathway and actin-cytoskeleton linked proteins in mediating the radiation injury response in spleen. Furthermore, we show that 24 h pretreatment with GT3 attenuates radiation-induced hematopoietic injury in the spleen by modulating various cell signaling proteins. Taken together, our results show that the radioprotective effects of GT3 are mediated, via alleviation of radiation-induced alterations in biochemical pathways, with wide implications on overall hematopoietic injury.

PEGylated IL-11 (BBT-059): A Novel Radiation Countermeasure for Hematopoietic Acute Radiation Syndrome.

Interleukin-11 was developed to reduce chemotherapy-induced thrombocytopenia; however, its clinical use was limited by severe adverse effects in humans. PEGylated interleukin-11 (BBT-059), developed by Bolder Biotechnology, Inc., exhibited a longer half-life in rodents and induced longer-lasting increases in hematopoietic cells than interleukin-11. A single dose of 1.2 mg kg of BBT-059, administered subcutaneously to CD2F1 mice (12-14 wk, male) was found to be safe in a 14 d toxicity study. The drug demonstrated its efficacy both as a prophylactic countermeasure and a mitigator in CD2F1 mice exposed to Co gamma total-body irradiation. A single dose of 0.3 mg kg, administered either 24 h pre-, 4 h post-, or 24 h postirradiation increased the survival of mice to 70-100% from lethal doses of radiation. Preadministration (-24 h) of the drug conferred a significantly (p < 0.05) higher survival compared to 24 h post-total-body irradiation. There was significantly accelerated recovery from radiation-induced peripheral blood neutropenia and thrombocytopenia in animals pretreated with BBT-059. The drug also increased bone marrow cellularity and megakaryocytes and accelerated multilineage hematopoietic recovery. In addition, BBT-059 inhibited the induction of radiation-induced hematopoietic biomarkers, thrombopoietin, erythropoietin, and Flt-3 ligand. These results indicate that BBT-059 is a promising radiation countermeasure, demonstrating its potential to be used both pre- and postirradiation for hematopoietic acute radiation syndrome with a broad window for medical management in a radiological or nuclear event.

Impact of Abbreviated Filgrastim Schedule on Survival and Hematopoietic Recovery after Irradiation in Four Mouse Strains with Different Radiosensitivity.

Filgrastim (Neupogen®, granulocyte-colony stimulating factor) is among the few countermeasures recommended for management of patients in the event of lethal total-body irradiation. Despite the plethora of studies using filgrastim as a radiation countermeasure, relatively little is known about the optimal dose schedule of filgrastim to mitigate radiation lethality. We evaluated the efficacy of filgrastim in improving 30-day survival of CD2F1 mice irradiated with a lethal dose (LD70/30) in the AFRRI cobalt-60 facility. We tested different schedules of 1, 3, 5, 10 or 16 once-daily injections of filgrastim initiated one day after irradiation. Time optimization studies with filgrastim treatment were also performed, beginning 6-48 h postirradiation. Maximum survival was observed with 3 daily doses of 0.17 mg/kg filgrastim. Survival efficacy of the 3-day treatment was compared against the conventional 16-day filgrastim treatment after irradiation in four mouse strains with varying radiation sensitivities: C3H/HeN, C57BL/6, B6C3F1 and CD2F1. Blood indices, bone marrow histopathology and colony forming unit assays were also evaluated. Filgrastim significantly increased 30-day survival (P < 0.001) with a 3-day treatment compared to 16-day treatment. Filgrastim did not prevent cytopenia nadirs, but facilitated faster recovery of white blood cells, neutrophils, red blood cells, platelets, lymphocytes and hematocrits in all four strains. Accelerated hematopoietic recovery was also reflected in faster bone marrow reconstitution and significant increase in hematopoietic progenitors (P < 0.001) in all four mouse strains. These data indicate that prompt and abbreviated filgrastim treatment has potential benefit for triage in the event of a radiological incident for treating acute hematopoietic syndrome.

Development of Orally Administered γ-Tocotrienol (GT3) Nanoemulsion for Radioprotection.

The purpose of this study was two-fold: (1) to formulate γ-tocotrienol (GT3) in a nanoemulsion formulation as a prophylactic orally administered radioprotective agent; and (2) to optimize the storage conditions to preserve the structural integrity of both the formulation and the compound. γ-tocotrienol was incorporated into a nanoemulsion and lyophilized with lactose. Ultra performance liquid chromatography-mass spectroscopy (UPLC-MS) was used to monitor the chemical stability of GT3 over time, the particle size and ζ potential, and scanning electron microscopy (SEM) were used to study the physical stability of the nanoemulsion. Radioprotective and toxicity studies were performed in mice. The liquid formulation exhibited GT3 degradation at all storage temperatures. Lyophilization, in the presence of lactose, significantly reduced GT3 degradation. Both the liquid and lyophilized nanoemulsions had stable particle size and ζ potential when stored at 4 °C. Toxicity studies of the nanoemulsion resulted in no observable toxicity in mice at an oral dose of 600 mg/kg GT3. The nano-formulated GT3 (300 mg/kg) demonstrated enhanced survival efficacy compared to GT3 alone (200 and 400 mg/kg) in CD2F1 mice exposed to total body gamma radiation. The optimal long-term storage of formulated GT3 is as a powder at -20 °C to preserve drug and formulation integrity. Formulation of GT3 as a nanoemulsion for oral delivery as a prophylactic radioprotectant shows promise and warrants further investigation.

Gamma-Tocotrienol Modulates Radiation-Induced MicroRNA Expression in Mouse Spleen.

Ionizing radiation causes depletion of hematopoietic cells and enhances the risk of developing secondary hematopoietic malignancies. Vitamin E analog gamma-tocotrienol (GT3), which has anticancer properties, promotes postirradiation hematopoietic cell recovery by enhancing spleen colony-forming capacity, and provides protection against radiation-induced lethality in mice. However, the underlying molecular mechanism involved in GT3-mediated postirradiation survival is not clearly understood. Recent studies have shown that natural dietary products including vitamin E provide a benefit to biological systems by modulating microRNA (miR) expression. In this study, we show that GT3 differentially modulates the miR footprint in the spleen of irradiated mice compared to controls at early times (day 1), as well as later times (day 4 and 15) after total-body irradiation. We observed that miR expression was altered in a dose- and time-dependent manner in GT3-pretreated spleen tissues from total-body irradiated mice. GT3 appeared to affect the expression of a number of radiation-modulated miRs known to be involved in hematopoiesis and lymphogenesis. Moreover, GT3 pretreatment also suppressed the upregulation of radiation-induced p53, suggesting the function of GT3 in the prevention of radiation-induced damage to the spleen. In addition, we have shown that GT3 significantly reduced serum levels of Flt3L, a biomarker of radiation-induced bone marrow aplasia. Further in silico analyses of the effect of GT3 implied the association of p38 MAPK, ERK and insulin signaling pathways. Our study provides initial insight into the mechanism by which GT3 mediates protection of spleen after total-body irradiation.